Methods for prevention or amelioration of West Nile encephalitis using a West Nile DNA vaccine

ABSTRACT

The present invention provides a safe and effective vaccine composition against West Nile virus disease. An immunogenically active component of West Nile virus or plasmid DNA, an adjuvant such as a metabolizable oil, and a pharmacologically acceptable carrier are formulated into an immunizing vaccine. The invention also provides a method for the prevention or amelioration of West Nile disease, such as encephalitis, in equidae by administering the vaccine composition herein set forth.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/202,716, filed on Jul. 25, 2002, now U.S. Pat. No. 7,153,513, whichclaims priority from U.S. application Ser. No. 60/308,334 filed on Jul.27, 2001.

FIELD OF THE INVENTION

The present invention relates to safe and effective West Nile Virusvaccines, and to methods of administering same to mammals, in particularhorses.

BACKGROUND OF THE INVENTION

Known as a Flavivirus, the West Nile virus was first identified in 1937in Africa and first found in North America in 1999. Migratory birds areconsidered the primary means whereby infection is spread within andbetween countries. The virus is transmitted by mosquitoes that haveacquired infection by feeding on viremic birds. The virus is thenamplified during periods of adult mosquito blood-feeding. Infectedmosquitos then transmit the virus to humans and animals upon feedingthereon.

West Nile virus is the causative agent for West Nile Virus disease,particularly West Nile encephalitis, predominately in humans, othermammals and birds. The chief concern in both the United States andforeign countries is the lack of effective treatment for West Nile virusdisease. Anti-inflammatory drugs are used to combat swelling of centralnervous system tissues, but beyond that no medical intervention isavailable. Nor is there believed to be a suitable vaccine known toprevent the infection. To date, preventing contact with carriers appearsto be the only means of controlling the West Nile virus.

What is therefore needed in the art is to provide a safe and effectiveequine West Nile virus vaccine composition. The vaccine compositionshould be sufficiently safened so as to be suitable for administrationeven to pregnant mares without adverse effect.

Also needed is a method for the prevention or amelioration of West NileVirus disease, particularly West Nile encephalitis, in equidae and othermammals.

SUMMARY OF THE INVENTION

The present invention provides a safened vaccine composition whichcomprises: an effective immunizing amount of an immunogenically activecomponent selected from the group consisting of an inactivated whole orsubunit West Nile virus, an antigen derived from said virus, DNA derivedfrom said virus, and a mixture thereof; an immunogenically stimulatingamount of a metabolizable oil; and a pharmacologically acceptablecarrier.

The present invention also provides a method for the prevention oramelioration of West Nile encephalitis in equidae which comprisesadministering to said equidae a safened vaccine composition whichcomprises an effective immunizing amount of an immunogenically activecomponent selected from the group consisting of an inactivated whole orsubunit West Nile virus, an antigen derived from said virus, DNA derivedfrom said virus, and a mixture thereof; an immunogenically stimulatingamount of a metabolizable oil; and a pharmacologically acceptablecarrier.

Also provided as part of the invention is a safe and effective West NileVirus vaccine suitable for use in horses which comprises animmunogenically active component selected from the group consisting ofan inactivated whole or subunit West Nile virus, an antigen derived fromsaid virus, DNA derived from said virus, and a mixture thereof; animmunogenically stimulating amount of a metabolizable oil; and apharmacologically acceptable carrier.

In a further embodiment, the invention describes a vaccine compositioncomprising at least about at least about 1×10⁴ TCID₅₀ per unit dose ofinactivated West Nile virus, or a component thereof, and about 4% to 10%vol/vol of a metabolizable oil comprising about 1 to 3%polyoxyethylene-polyoxypropylene block copolymer, about 2 to 6% ofsqualane and about 0.1 to 0.5% of polyoxyethylene sorbitan monooleate.

Further provided is a safened and effective West Nile virus vaccine forequidae, comprising at least about 1×10⁶ TCID₅₀ per unit dose of killedor inactivated West Nile virus, and at least about 1% vol/vol of anadjuvant comprising at least one metabolizable oil and at least onewetting or dispersing agent.

The invention also sets forth a vaccine regimen for horses, comprisingtwo dosage units of killed or inactivated West Nile virus, wherein eachsaid dosage unit comprises about 0.5 to 5 milliliters of a compositioncontaining at least about 5×10⁷ TCID₅₀ of said virus and about 1 to 10%vol/vol of an adjuvant, said adjuvant comprising at least onemetabolizable oil and at least two nonionic surfactants, and furtherwherein said dosage unit comprises a pharmacologically acceptablecarrier.

Also provided as part of the invention is an equine vaccine containingWest Nile plasmid DNA in an amount of about 50 to 3,000 micrograms perdose, together with one or more adjuvants and a suitable carrier. Avaccine regimen would comprise administering at least about one dose ofthis composition, and desirably at least about 2 doses, to horses forimmunization against West Nile virus disease.

Further, objects and features of the invention will become apparent fromthe detailed description and the claims set forth herein below.

DETAILED DESCRIPTION OF THE INVENTION

Scientists believe that the West Nile virus follows the same pattern ofinfection found with other mosquito-transmitted viruses. When aninfected mosquito bites an equid, the virus enters the skin or tissuesjust below the bite site, where it is picked up by the circulation. Thevirus can multiply in the bloodstream, and the equid may develop afever, which often goes undetected because there are no other signs ofillness at the time. However, once the virus has invaded the nervoussystem, clinical signs appear within one to three days. Most affectedequidae, such as horses, first exhibit signs of posterior weakness orparalysis and poor coordination. Depression and related behavior changesmay accompany the physical changes. In severe cases, tremors,convulsions, paddling of the limbs and paralysis may develop. Severeneurological problems and mortality have also been observed. To date, novaccine known to prevent West Nile virus in equidae is available; andthe only means of controlling the West Nile virus appears to be theprevention of contact with a carrier.

It has now been found that a safe and effective vaccine compositionwhich comprises an effective immunizing amount of an immunogenicallyactive component selected from the group consisting of an inactivatedwhole or subunit West Nile virus, an antigen derived from said virus,DNA derived from said virus, plasmid West Nile virus DNA, plasmid withsequence inserts of said virus, and a mixture thereof; animmunogenically stimulating amount of an adjuvant, in particular ametabolizable oil; and a pharmacologically acceptable carrier may beadministered to equidae, particularly horses, to prevent or ameliorateWest Nile Virus disease such as encephalitis.

DNA derived from the West Nile virus may be obtained via isolation fromsources such as the fluids or tissues of equine or avian speciesdiagnosed to have West Nile encephalitis. Such sources include cerebralspinal fluid or sections of spinal cord or brain. DNA may also beobtained using other available techniques such as plasmid technology.For example, suitable cells of an organism, e.g. E. coli, may betransformed with a plasmid containing West Nile protein sequence insertsto obtain a master seed. The master seed may then be cultured andpassaged. Transformed cells containing the West Nile DNA may then beharvested, and the DNA isolated and obtained using techniques availableto the skilled artisan.

Whole or subunit West Nile virus may be isolated from infected animalsusing conventional techniques. Samples of the virus may also be obtainedfrom tissue culture collections which maintain a depository fororganisms such as West Nile. At the American Type Culture Collection(ATCC), for example, the West Nile virus has been deposited under ATCCNo.s VR-82,VR-1267 and VR-1267 AF.

Whole or subunit West Nile virus may be inactivated by conventionalinactivating means, for example chemical inactivation using chemicalinactivating agents such as binary ethyleneimine, beta-propiolactone,formalin, gluteraldehyde, sodium dodecyl sulfate, or the like or amixture thereof, preferably formalin. Said virus may also be inactivatedby heat or psoralen in the presence of ultraviolet light. (Live,attenuated West Nile virus may also be used in certain embodiments, butthis is perhaps much less preferred.)

Whole or subunit West Nile virus may be maintained or grown in mousebrains or in suitable tissue culture media, such as optiMEM (LTI, GrandIsland, N.Y.) or MEM media, or in cells known in the art such as Africangreen monkey kidney (Vero) cells or baby hamster (BHK) cells, preferablyVero cells. Said virus may then be separated from the tissue culture orcell media using conventional techniques such as centrifugation,filtration or the like.

A preferred West Nile virus isolate may be obtained from the NationalVeterinary Services Laboratory (part of the United States Department ofAgriculture) in Ames, Iowa as strain VM-2. The virus strain may beplaque purified up to three times, and passaged to X+5 in Vero cells.

As used herein the term “immunogenically active” designates the abilityto stimulate an immune response, i.e., to stimulate the production ofantibodies, particularly humoral antibodies, or to stimulate acell-mediated response. For example, the ability to stimulate theproduction of circulating or secretory antibodies or the production of acell-mediated response in local mucosal regions, (e.g., intestinalmucosa), peripheral blood, cerebral spinal fluid or the like.

The effective immunizing amount of the immunogenically active componentmay vary and may be any amount sufficient to evoke an immune responseand provide immunological protection against West Nile Virus disease.Amounts wherein a dosage unit comprises at least about 1×10⁴ TCID₅₀ ofkilled or inactivated whole or subunit virus cells or antigen or DNAcells derived therefrom or a mixture thereof, preferably at least about1×10⁶ TCID₅₀, are suitable. Even more preferably, at least about 1×10⁷TCID₅₀ per dosage unit may be utilized. It is especially desirable thatat least about 5×10⁷ TCID₅₀ of killed or inactivated whole or subunitWest Nile virus cells or antigen or DNA cells derived therefrom or amixture thereof be used in the vaccine composition of the invention. Incertain embodiments, as much as 1×10⁹ TCID₅₀ and more may be utilized. Arange of about 1×10⁴ TCID₅₀ to about 1×10⁸ TCID₅₀ may also be utilized.

In a further embodiment of the invention, it is contemplated that about50 to 3,000 micrograms (μg or 10⁻⁶ grams) of West Nile plasmid DNA maybe utilized in one dosage unit of the vaccine composition. Morepreferably, about 100 to 1,000 μg may be used, with about 100 to 250 μgof plasmid DNA being more preferred.

At least one dosage unit per animal is contemplated herein as avaccination regimen. In some embodiments, two or more dosage units maybe especially useful. A dosage unit may typically be about 0.1 to 10milliliters of vaccine composition, preferably about 0.5 to 5milliliters, and even more preferably about 1 to 2 milliliters, witheach dosage unit containing the heretofore described quantity of virusor virus component. The skilled artisan will quickly recognize that aparticular quantity of vaccine composition per dosage unit, as well asthe total number of dosage units per vaccination regimen, may beoptimized, so long as an effective immunizing amount of the virus or acomponent thereof is ultimately delivered to the animal.

The West Nile virus vaccine composition of the invention may alsocontain one or more adjuvants. As used herein the term “adjuvant” refersto any component which improves the body's response to a vaccine. Theadjuvant will typically comprise about 0.1 to 50% vol/vol of the vaccineformulation of the invention, more preferably about 1 to 50% of thevaccine, and even more desirably about 1 to 20% thereof. Amounts ofabout 4 to 10% may be even more preferred.

Suitable adjuvants can include immunostimulating oils such as certainmetabolizable oils. Metabolizable oils suitable for use in thecomposition of the invention include oil emulsions, e.g., SP oil(hereinafter described), Emulsigen (MPV Laboratories, Ralston, NZ),Montanide 264,266,26 (Seppic SA, Paris, France), as well as peanut oiland other vegetable-based oils, squalane (shark liver oil) or othermetabolizable oil which can be shown to be suitable as an adjuvant inveterinary vaccine practice.

In addition, the adjuvant may include one or more wetting or dispersingagents in amounts of about 0.1 to 25%, more preferably about 1 to 10%,and even more preferably about 1 to 3% by volume of the adjuvant.Particularly preferred as wetting or dispersing agents are non-ionicsurfactants. Useful non-ionic surfactants includepolyoxyethylene/polyoxypropylene block copolymers, especially thosemarketed under the trademark PLURONIC® and available from BASFCorporation (Mt. Olive, N.J.). Other useful nonionic surfactants includepolyoxyethylene esters such as polyoxyethylene sorbitan monooleate,available under the trademark TWEEN 80®. It may be desirable to includemore than one, e.g. at least two, wetting or dispersing agents in theadjuvant as part of the vaccine composition of the invention.

Other components of the adjuvant may include such preservative compoundsas formalin and thimerosal in amounts of up to about 1% vol/vol of theadjuvant.

As an adjuvant, SP oil is preferred. As used in the specification andclaims, the term “SP oil” designates an oil emulsion comprising apolyoxyethylene-polyoxypropylene block copolymer, squalane,polyoxyethylene sorbitan monooleate and a buffered salt solution. Ingeneral, the SP oil emulsion will comprise about 1 to 3% vol/vol ofblock copolymer, about 2 to 6% vol/vol of squalane, more particularlyabout 3 to 6% of squalane, and about 0.1 to 0.5% vol/vol ofpolyoxyethylene sorbitan monooleate, with the remainder being a bufferedsalt solution.

When utilized, immunogenically stimulating amounts of SP oil as adjuvantin the vaccine composition of the invention may vary according to theimmunogenically active component, the degree of potential infectiousexposure, method of administration of the vaccine composition, the ageand size of the equid, or the like. In general, amounts of about 1% to50% vol/vol, preferably about 4% to 10% vol/vol, and more preferablyabout 4% to 5% vol/vol of SP oil are suitable.

In general, it is believed that a live virus vaccine may potentiallylack sufficient safety in a given target host, and that a killed orinactivated virus vaccine may potentially lack the ability to stimulatea sufficiently effective immunologic response. Commonly, an adjuvant orimmunogenically stimulating compound is used in combination with akilled or inactivated virus in a vaccine composition to obtainacceptable efficacy. However, safety to the target host is oftencompromised by the addition of an adjuvant. For example, pregnantanimals many times have been known to have a significantly higher rateof miscarriage after being administered a killed or inactivated virusvaccine that contains an adjuvant.

It has now been found that when a suitable adjuvant, e.g. ametabolizable oil preferably such as SP oil, is used in combination withan immunogenically active component as described hereinabove, theresultant West Nile vaccine composition is safened for use in equidae,particularly horses, even for use in pregnant mares, while demonstratingimportant efficacy as well. Thus, the invention achieves the concomitantgoals of effective immunization and safety, especially for pregnantanimals. This combination of active immunogen and adjuvant is unheraldedin the art.

Pharmacologically acceptable carriers suitable for use in the vaccinecomposition of the invention may be any conventional liquid carriersuitable for veterinary pharmaceutical compositions, preferably abalanced salt solution or other water-based solution suitable for use intissue culture media. Other available carriers may also be utilized.

Additional excipients available in the art may also be Included in thevaccine composition according to the various embodiments heretoforedescribed. For example, pH modifiers may be utilized.

The components of the vaccine composition of the invention as heretoforedescribed, including the carrier, may be combined together usingavailable techniques.

In addition to the immunogenically active component of West Nile virusas described hereinabove as active ingredient, it is contemplated thatthe vaccine composition of the invention may also contain other activecomponents such as an antipathogenic component directed against rabiesvirus, Eastern equine encephalitis virus, Western equine encephalitisvirus, Venezuelan equine encephalitis virus, equine herpes virus such asEHV-1 or EHV-4, Ehrlichia risticii, Streptococcus equi, tetanus toxoid,equine influenza virus (EIV), or the like or a combination thereof. Thequantities of one or more of these viruses may be determined fromefficacy literature in the art, or determined using availabletechniques.

In one embodiment of the invention the immunogenically active componentof the invention may be incorporated into liposomes using knowntechnology such as that described in Nature, 1974, 252, 252-254 orJournal of Immunology, 1978, 120, 1109-13. In another embodiment of theinvention, the immunogenically active component of the invention may beconjugated to suitable biological compounds such as polysaccharides,peptides, proteins, or the like, or a combination thereof.

In a preferred embodiment of the invention, the inventive vaccinecomposition may be formulated in dosage unit form as heretoforedescribed to facilitate administration and ensure uniformity of dosage.Formulation may be effected using available techniques, such as thoseapplicable to preparations of emulsions.

The inventive vaccine composition may be administered parenterally, forexample, intramuscularly, subcutaneously, intraperitoneally,intradermally or the like, preferably intramuscularly; or saidcomposition may be administered orally or intranasally.

Accordingly, the present invention also provides a method for theprevention or amelioration of West Nile encephalitis in equidae,preferably horses, which comprises administering to said equidae asafened vaccine composition as described hereinabove.

In actual practice, the vaccine composition of the invention isadministered parenterally, subcutaneously, orally, intranasally, or byother available means, preferably parenterally, more preferablyintramuscularly, in effective amounts according to a schedule which maybe determined by the time of anticipated potential exposure to a carrierof the West Nile virus. In this way, the treated animal may have time tobuild immunity prior to the natural exposure. By way of non-limitingexample, a typical treatment schedule or dosing regimen may includeparenteral administration, preferably intramuscular injection of onedosage unit, at least about 2-8 weeks prior to potential exposure. Atleast two administrations are preferred, for example one dosage unit atabout 8 weeks and a second dosage unit at about 3-5 weeks prior topotential exposure of the treated animal. As heretofore set forth, adosage unit will typically be within the range of about 0.1 to 10milliliters of vaccine composition containing the previously describedamounts of active and percentages of adjuvant and inactive(s) as setforth. A dosage unit within the range of about 0.5 to 5 milliliters isperhaps more preferred, with about 1 to 2 milliliter(s) beingparticularly preferred.

For a clearer understanding of the invention, the following examples areset forth below. These examples are merely illustrative and are notunderstood to limit the scope or underlying principles of the inventionin any way. Indeed, various modifications of the invention, in additionto those shown and described herein, will become apparent to thoseskilled in the art from the following examples and the foregoingdescription. Such modifications are also intended to fall within thescope of the appended claims.

EXAMPLE 1 Preparation of Vaccine

A/Formulation of SP Oil

Ingredient Description Volume Polyoxyethylene-polyoxypropylene blockcopolymer 20.0 ml (Pluronic ® L121, BASF, Mt. Olive, NJ) Squalane(Kodak, Rochester, NY) 40.0 ml Polyoxyethylenesorbitan monooleate  3.2ml (Tween ® 80, Sigma Chemical, St. Louis, MO) Buffered salt solution936.8 ml  (D-V PBS Solution, Ca, Mg free)

The ingredients are mixed and homogenized until a stable mass oremulsion is formed. Prior to homogenization, the ingredients or mixturemay be autoclaved. The emulsion may be further sterilized by filtration.Formalin may be added up to a final concentration of 0.2%. Thimerosalmay be added to a final dilution of 1:10,000.

B/Vaccine Preparation

An equine isolate of West Nile virus, obtained from USDA facilities inAmes, Iowa (Lot No. VM-2,Equine Origin, 1999 North American isolate,second passage in VeroM cell culture), was cultivated in multiplecultures of Vero cells in OptiMEM (LTI, Grand Island, N.Y.) tissueculture medium at 37° C. The harvests are titrated and then inactivatedby means of addition of a 10% formalin solution to a final concentrationof 0.1%. This is allowed to inactivate at 37° C. for a period of no lessthan 144 hours. Then, another addition of 0.1% formalin is added andincubated at 37° C. for another period of no less than 144 hours.

The vaccines are formulated by suspending the appropriate volume ofinactivated virus fluid in 1-20% by volume of SP oil per 1 mL dose.

EXAMPLE 2 Evaluation of Antibody Response to the Intramuscular Injectionof Test Vaccine

In this evaluation, horses are randomly divided into four groups: onegroup of twenty horses is administered test vaccine at a dose of 1×10⁷TCID₅₀ (Tissue Culture Infectious Dose); a second group of twenty horsesis administered test vaccine at a dose of 5×10⁷ TCID₅₀; a third group offive horses is administered test vaccine at a dose of 1×10⁸ TCID₅₀; anda fourth group of eight horses are maintained as non-vaccinatedenvironmental controls. Treated horses are given a first dose of vaccineaccording to the group to which they are assigned. At twenty-one daysfollowing administration of the first dose, a second dose of the samevaccine is administered. All horses are bled for serum at the time ofadministration of the first and second dose and at weekly intervalsthrough 28 days post second dose administration.

Test Vaccine A Component Conc./Dose Volume/mL West Nilevirus-Inactivated 1 × 10⁷ TCID₅₀ 0.0347 mL MEM¹ NA 0.9138 mL SP oil 5%0.0500 mL Polymyxin B² 30.0 mcg/mL 0.0003 mL Neomycin 30.0 mcg/mL 0.0003mL Thimerosal (5%) 1:20,000 0.0010 mL

Test Vaccine B Component Conc./Dose Volume/mL West Nilevirus-Inactivated 5 × 10⁷ TCID₅₀ 0.1734 mL MEM NA 0.7752 mL SP oil 5%0.0500 mL Polymyxin B² 30.0 mcg/mL 0.0002 mL Neomycin³ 30.0 mcg/mL0.0002 mL Thimerosal⁴ (5%) 1:20,000 0.0010 mL

Test Vaccine C Component Conc./Dose Volume/mL West Nilevirus-Inactivated 1 × 10⁸ TCID₅₀ 0.3467 mL MEM¹ NA 0.6019 mL SP oil 5%0.0500 mL Polymyxin B² 30.0 mcg/mL 0.0002 mL Neomycin³ 30.0 mcg/mL0.0002 mL Thimerosal⁴ (5%) 1:20,000 0.0010 mL ¹LTI, Grand Island, NY²Sigma, St. Louis, MO ³Sigma, St. Louis, MO ⁴Sigma, St. Louis, MO

The serological data obtained is shown in Table I below, wherein: 0 DPV1 designates day zero, pre vaccination; and 14 DPV 2 designates day 14,post vaccination. NR designates no results.

As can be seen from the data on Table I, treated horses from all groupsshowed significant increases in antibodies to West Nile virus while thecontrol horses maintained a non-detectable antibody level. The level ofresponse in the horses that received vaccine was independent of thelevel of antigen in the vaccine that they received.

TABLE I Test Dose Test #1 Test #2 Vaccine (TC1D₅₀) 0DPV1 14DPV2 0DPV114DPV2 A 1 × 10⁷ <10 160 <10 80 A 1 × 10⁷ <10 20 <10 10 A 1 × 10⁷ <10≧320 <10 40 A 1 × 10⁷ <10 80 <10 ≧320 A 1 × 10⁷ <10 80 <10 40 A 1 × 10⁷<10 80 <10 160 A 1 × 10⁷ <10 40 <10 40 A 1 × 10⁷ <10 80 <10 80 A 1 × 10⁷<10 160 <10 80 A 1 × 10⁷ <10 40 <10 10 A 1 × 10⁷ <10 40 <10 10 A 1 × 10⁷<10 160 <10 40 A 1 × 10⁷ <10 ≧320 <10  NR* A 1 × 10⁷ <10 ≧320 <10 NR A 1× 10⁷ <10 ≧320 <10 80 A 1 × 10⁷ <10 160 <10 20 A 1 × 10⁷ ≧20 ≧320 <10160 A 1 × 10⁷ <10 80 <10 80 A 1 × 10⁷ <10 80 <10 160 A 1 × 10⁷ <10 160<10 160 B 5 × 10⁷ <10 80 <10 40 B 5 × 10⁷ <10 20 <10 <10 B 5 × 10⁷ <10≧320 <10 160 B 5 × 10⁷ <10 80 <10 ≧320 B 5 × 10⁷ <10 ≧320 <10 160 B 5 ×10⁷ <10 80 <10 80 B 5 × 10⁷ <10 160 <10 ≧320 B 5 × 10⁷ <10 80 <10 40 B 5× 10⁷ <10 160 <10 40 B 5 × 10⁷ <10 40 <10 80 B 5 × 10⁷ <10 20 <10 20 B 5× 10⁷ <10 ≧320 <10 160 B 5 × 10⁷ <10 ≧40 <10 ≧320 B 5 × 10⁷ <10 80 <1040 B 5 × 10⁷ <10 80 <10 NR B 5 × 10⁷ <20 ≧320 <10 80 B 5 × 10⁷ ≧20 160<10 80 B 5 × 10⁷ <10 ≧320 <10 160 B 5 × 10⁷ <10 ≧320 <10 ≧320 B 5 × 10⁷<10 80 <10 160 C 1 × 10⁸ <10 ≧320 <10 ≧320 C 1 × 10⁸ <10 ≧320 <10 160 C1 × 10⁸ <10 160 <10 80 C 1 × 10⁸ <10 ≧320 <10 NR C 1 × 10⁸ <10 40 <10 40Control 0 <10 <10 <10 <10 Control 0 <10 <10 <10 <10 Control 0 <10 <10<10 <10 Control 0 <10 <10 <10 NR Control 0 <10 <10 <10 <10 Control 0 <10<10 <10 <10 Control 0 <10 <10 <10 <10 Control 0 <10 <10 <10 <10

EXAMPLE 3 Evaluation of Safety of Test Vaccine in Horses Under FieldConditions

In this evaluation, 648 healthy male and female horses, including 32pregnant mares, are vaccinated with a 6×10⁶ TCID₅₀ dose of inactivatedtest vaccine administered as a 1 mL dose vaccination by intramuscularadministration and followed in three to four weeks by a second 1 mL dosevaccination. The treated horses are housed and fed using conventionalhusbandry practices for farm or stable. All treated horses are observedby a veterinarian for 30 minutes following vaccination for immediatereactions such as salivation, labored or irregular breathing, shaking,or anaphylaxis. For two weeks post-vaccination, the horses are observeddaily for any delayed reactions such as lethargy, anorexia or unusualswelling at the injection site. Blood samples of 5 to 10 mL are taken byvenipuncture from treated horses on the day of first vaccination (dayZero) and at least once more at two or more weeks post secondvaccination (day 36 or greater). Serological assays using PRNT⁵ testingare performed. ⁵Chang, G. J.; Hunt, A. R. and Davis B., Journal ofVirology, 74 pp 4244-5422 (2000).

Test Vaccine Component Conc./Dose Volume/mL West Nile virus-Inactivated6 × 10⁸ TCID₅₀ 0.21 mL SP oil 5% 0.05 mL MEM N/A 0.74 mL

No vaccine-induced safety problems were found in any of the vaccinates,including the pregnant mares. This evaluation demonstrates that thevaccine of the invention is safe for use in horses under fieldconditions.

EXAMPLE 4 Evaluation of Efficacy of Test Vaccine (Multivalent andMonovalent Preparations) in Horses Under Experimental Conditions

The efficacy of a combination vaccine containing killed West Nile virus(WNV) against experimental WNV challenge was evaluated.

A total of 30 horses were allotted into one vaccinated group (20 horses)and one control group (10 horses). Horses in the vaccinated groupreceived intramuscularly two doses of the test vaccine containing killedWest Nile virus (5×10⁷ TCID₅₀ per dose with 5% SP oil), influenza virus,encephalomyelitis virus (Eastern, Western and Venezuelan),rhinopneumonitis virus (serotypes 1 and 4), and tetanus toxoid, threeweeks apart. Serum samples were collected periodically for antibodyresponse measured by plaque reduction neutralization test (PRNT).Twenty-four (24) days after the second vaccination, all horses werechallenged subcutaneously with WNV. After challenge, horses weremonitored for rectal temperature and any clinical signs twice daily fortwo weeks and once weekly thereafter for detection of viremia. Horseswere euthanized and necropsied on 21 and 22 DPC. Cerebrospinal fluid(CSF), spinal cord (cervical, thoracic, and lumbar) and brain (frontal,occipital, medulla oblongata, and brain stem) tissue samples wereexamined for gross pathology and collected for virus isolation.

Fourteen days after the second vaccination, 75% of vaccinated animalsseroconverted (titer≧5) with a geometric mean titer of 10 while controlanimals remained negative (titer<5). The vaccination conferred asignificant protection against viremia (a precursor to development offull-blown West Nile Virus disease). Nine out of 10 (90%) controlsdeveloped viremia after challenge, while only eight out of 20 (40%)vaccinates had transient viremia, or viremia lasting only a few days atmost. Importantly, no WNV disease clinical signs were observed in any ofthe challenged vaccinated animals throughout the observation period.(Transient febrile responses were observed in one control and twovaccinated horses. However, there was no evidence to suggest theseresponses were due to WNV infection.) Petechial hemorrhage in whitematter and subdural hemorrhage were found in the brain tissue from onecontrol animal. WNV was isolated from the brain but not from CSF andspinal cord samples collected from this animal. No WNV was isolated fromany of the tissue samples collected from other challenged horses.

Results from this study demonstrated a significant protection againstboth viremia and signs of clinical WNV disease in horses vaccinated withthe test combination vaccine.

A second study has conducted with a protocol similar to that above,except that a monovalent WNV vaccine (WNV vaccine alone) was utilized,and all horses were challenged with WNV at 12 months after the secondvaccination. Nine out of 11 (81.8%) of the controls developed viremiaafter challenge, while only one out of 19 (5.3%) of the vaccinates hadtransient viremia. No WNV associated clinical signs were observed in anyof the challenged animals throughout the observation period. No febrileresponses were observed in any of the challenged horses. No WNV wasisolated from any of the tissue or CSF samples collected from any of thechallenged horses. (Prior to challenge at the end of the 12 monthperiod, 17 of the nineteen vaccinated horses had plaque reductionneutralization test (PRNT) titers of 5 or greater, while the controlgroup remained negative (<5).)

Results from this second study demonstrate a significant protection (94%of preventable fraction) against viremia in horses vaccinated with thekilled monovalent WNV vaccine. These results also demonstrate a longduration of protective immunity.

EXAMPLE 5 Evaluation of Efficacy of DNA Test Vaccine in Horses UnderExperimental Conditions

This example demonstrates the efficacy of a West Nile Virus (WNV) DNAvaccine, as part of a further embodiment of the invention. The DNAvaccine contained 100 μg of purified DNA adjuvanted with 5% SP oil per 2mL dose, and was evaluated against experimental WNV challenge.

For the composition of the WNV DNA vaccine, bacterial cells wereharvested from a culture passaged 10 times from a master seed using E.coli DH10B obtained from Invitrogen (Carlsbad, Calif.) containing a WestNile plasmid pCBWN obtained from the Centers for Disease Control (FortCollins, Colo.). The bacterial cells were suspended in glucose-tris-EDTAbuffer and lysed with sodium hydroxide and sodium dodecyl sulfate. Thelysate was neutralized with a potassium acetate solution. Theprecipitated complex material containing DNA, RNA, cell debris andproteins was removed by filtration. The filtrate was precipitated withthe addition of isopropyl alcohol. The precipitate was collected bycentrifugation and resuspended in buffer. This process was repeatedusing ammonium acetate. The precipitate collected was resuspended inbuffer and loaded into a chromatography column packed with Polyflo®resin. The column was then washed and the plasmid DNA was eluted fromthe column. The eluate was finally diafiltered extensively againstphosphate buffer saline. The purified plasmid DNA stocks were thenshipped for blending. The test vaccine contained 100 μg of plasmid DNAadjuvanted with 5% SP oil.

The horses used for testing were randomly assigned into two groups: 20animals received the WNV DNA vaccine, and 10 animals were used ascontrols. The first group were vaccinated intramuscularly with two 2.0mL doses of vaccine three weeks apart. The control horses received novaccinations or placebos. One group of horses (9 vaccinates and 5controls) were challenged 5 weeks after the second vaccination, whereasa second group of horses (11 vaccinates and 5 controls) were challenged12 weeks after the second vaccination. Briefly, Aedes albopictusmosquitoes which had been infected with WNV 12 days prior to the horsechallenge, were allowed to feed on each horse for at least 5 minutes.Following challenge, only those mosquitoes which were found to haveengorged with blood meals from each horse were frozen at −20° C., andthe virus load was titrated as a pool subsequently. Mosquitoes were thenhomogenized by vortexing using diluent. The homogenate was centrifugedand the supernatant was removed for titration on Vero cells.

After challenge, rectal temperatures were taken twice daily for at least9 days and clinical signs were monitored twice a day for at least 21days. Serum samples were taken twice a day for the first 9 days postchallenge (DPC); once daily from 10 to 14 DPC and finally at 21 DPC.Virus isolation was performed on serum samples from 0 DPC to 10 DPC forthe first challenge group and from 0 DPC to 11 DPC for the secondchallenge group. The first group of 14 horses and the second group of 16horses were euthanized and necropsied 28 to 37 DPC and 29 to 38 DPC,respectively. Cerebrospinal fluid (CSF) and tissue samples from thecerebrum, cerebellum and the brain stem were collected for grosspathology and virus isolation.

At 14 days post second vaccination, 6 of 20 vaccinated had a measurabletiter of 2 or greater, and one horse had a titer of 5. Vaccination hadconferred protection against experimental West Nile challenge usingmosquitoes. Viremia was detected in 5 of 5 control animals and 4 of 9vaccinates in the first challenge group, whereas 4 of 5 controls and 2of 11 vaccinates were viremic in the second challenge group of horses.The viremia detected was transient, and occurred only within the firstsix days after challenge. As a whole, viremia was detected in 9 out of10 (90%) control horses, while only 6 out of 20 (30%) vaccinates weredetected with viremia.

No neurological signs attributable to WNV infection were observed in anyof the study horses throughout the challenge observation period. No WNVwas isolated from any of the tissue samples collected from any of thestudy horses. One horse from the first challenge group which waseuthanized on 7 DPC showed no gross or microscopic evidence ofencephalitis or meningitis.

Results from this study demonstrated a significant protection againstviremia in horses vaccinated with the test vaccine.

1. A method for the prevention or amelioration of West Nile encephalitisin equidae which comprises administering to said equidae a vaccinecomposition which comprises an effective immunizing amount of West Nileplasmid DNA, wherein said plasmid DNA is pCBWN; an immunogenicallystimulating amount of a metabolizable oil; and a pharmacologicallyacceptable carrier.
 2. The method according to claim 1 having saidvaccine composition wherein the metabolizable oil is SP oil.
 3. Themethod according to claim 1 wherein said equidae are horses.
 4. Themethod according to claim 3 wherein said horses are pregnant mares. 5.The method according to claim 1 wherein said vaccine composition isadministered parenterally.
 6. The method according to claim 1 whereinsaid vaccine composition is administered intramuscularly.
 7. The methodaccording to claim 1 wherein the metabolizable oil is SP oil and ispresent in an amount of about 5% vol/vol.
 8. A method for the preventionor amelioration of West Nile encephalitis in equidae, said methodcomprising administering to said equidae a vaccine compositioncomprising West Nile virus plasmid DNA, wherein said plasmid DNA ispCBWN, in an amount of about 50 to 3,000 micrograms per dose and animmunogenically stimulating amount of a metabolizable oil.
 9. The methodof claim 8, wherein said composition comprises about 100 to 1,000micrograms of said plasmid DNA per dose.
 10. The method of claim 9,wherein said composition comprises about 100 to 250 micrograms of saidplasmid DNA per dose.